Development of 6-arylcoumarins as nonsteroidal progesterone antagonists. Structure–activity relationships and fluorescence properties

Article abstract of DOI:10.1016/j.bmc.2016.09.020

Progesterone is involved in multiple physiological processes, including female reproduction, via binding to the progesterone receptor (PR). We have developed 6-arylcoumarins such as 5 and 6 as non-steroidal PR antagonists with receptor-binding-dependent f

Full text of DOI:10.1016/j.bmc.2016.09.020

Accepted Manuscript
Development of 6-Arylcoumarins as Nonsteroidal Progesterone Antagonists.
Structure-activity Relationships and Fluorescence Properties
Marie Kinoshita, Mai Negishi, Haruka Sakai, Tomoya Hirano, Shuichi Mori,
Shinya Fujii, Hiroyuki Kagechika, Aya Tanatani
PII:
S0968-0896(16)30708-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.020
BMC 13273
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 August 2016
8 September 2016
9 September 2016
Please cite this article as: Kinoshita, M., Negishi, M., Sakai, H., Hirano, T., Mori, S., Fujii, S., Kagechika, H.,
Tanatani, A., Development of 6-Arylcoumarins as Nonsteroidal Progesterone Antagonists. Structure-activity
Relationships and Fluorescence Properties, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.09.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development of 6-Arylcoumarins as Nonsteroidal Progesterone Antagonists.
Structure-activity Relationships and Fluorescence Properties
Marie Kinoshita,a Mai Negishi,a Haruka Sakai,a Tomoya Hirano,b Shuichi Mori,b Shinya Fujii,b,c
Hiroyuki Kagechika,b and Aya Tanatani,a, *
aDepartment of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112-8610, Japan
bSchool of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and
Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
cInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-0032, Japan
To whom correspondence should be addressed:
Aya Tanatani, E-mail: tanatani.aya @ocha.ac.jp; Tel/Fax: +81-3- 5978-2716
Abstract
Progesterone is involved in multiple physiological processes, including female reproduction, via
binding to the progesterone receptor (PR). We have developed 6-arylcoumarins such as 5 and 6 as
non-steroidal PR antagonists with receptor-binding-dependent fluorescence. In this study, we
investigated the structure-activity relationships and fluorescence properties of coumarin derivatives
bearing a heterocyclic aromatic moiety. Among these derivatives, 7c (IC50: 34 nM) and 10b (IC50: 24
nM) showed more potent PR-antagonistic activity than lead compounds 5 (IC50: 500 nM) and 6
(IC50: 65 nM) in alkaline phosphatase (AP) assay. Compound 9b showed solvent-dependent
fluorescence intensity, exhibiting strong fluorescence in the presence of PR LBD only in buffer
solution. On the other hand, 10b showed a solvent-dependent shift of the fluorescence maximum
wavelength in the presence of PR LBD. These results indicate that 6-arylcoumarin will be a useful
scaffold for PR antagonists and fluorescent probes targeting PR.
Keywords
progesterone, antagonist, fluorescent ligand, coumarin

1. Introduction
Progesterone (1, Fig. 1) has significant roles in various physiological processes, including uterine
cell proliferation and differentiation, the ovulation cycle, and mammary gland growth and
differentiation in females.1-4 Functions of progesterone (1) in the cardiovascular,5 immune,6 and
nervous systems7 have also been reported. These activities of progesterone (1) are mediated by the
progesterone receptor (PR), which is a ligand-dependent transcriptional factor belonging to the
nuclear receptor superfamily.8 Various synthetic PR agonists and antagonists have been developed
for clinical use.9,10 For example, PR antagonist mifepristone (2) has been clinically used as an
abortifacient.11 Recent studies suggested that PR antagonists might be effective in the treatment of
endometriosis,12 uterine leiomyoma,13 and breast cancer.14,15 However, PR antagonists with a
steroidal skeleton, such as 2, show cross-activity toward other steroid hormone receptors,16 and often
have significant adverse effects. Therefore, nonsteroidal PR-antagonists have been developed, such
as 3a and 4a.17-21 Interestingly, even minor structural modification can change the biological
functions of these compounds; for example, compounds 3b, 3c, and 4b acted as PR agonists, not PR
antagonists.
Figure 1. Structures of progesterone (1), mifepristone (2), and typical nonsteroidal PR ligands, 3
and 4.
We previously reported 6-arylcoumarins as novel nonsteroidal PR antagonists.22 Compound 5
exhibited potent PR antagonistic activity in alkaline phosphatase (AP) assay with T47D human
breast carcinoma cells (IC50: 0.50 M), and showed high binding affinity towards recombinant hPR
ligand-binding domain (LBD) (IC50: 1.42 M). Compound 5 also has possible application as a
fluorescent probe for PR, since its fluorescence is increased in the presence of PR LBD, though the
fluorescence quantum yield (0.744 in methylene chloride) was moderate and depended on the
solvent properties. Further, compound 5 has binding affinity for other steroid hormone receptors,

such as hGR (IC50: 3.54 M) and hAR (IC50: 10.0 M). Therefore, novel compounds with higher
fluorescence quantum yield and greater PR selectivity are needed for development of fluorescent
probes for PR. Among the 6-arylcoumarin derivatives synthesized here, we found that some
compounds bearing a pyrrole or thiophene, but not furane, as 6-aryl group, showed potent PR
antagonistic activity.20 For example, compound 6 showed more potent PR antagonistic activity (IC50:
0.065 M) than 5 in AP assay. Although the binding affinity of 6 to recombinant hPR LBD is very
low (IC50: 15.2 M), 6 did not bind to other steroid hormone receptors.
Figure 2. Structures of PR-antagonistic coumarin derivatives
From a consideration of our previous work on coumarin derivatives with PR antagonistic activity,
together with the structures of known PR ligands,17-21 we expected that linkage of coumarin to a
five-membered heterocyclic ring would be favorable for high PR-antagonistic activity. Further,
introduction of appropriate substituents on the coumarin ring, especially at the 7 position or on the
five-membered heterocyclic ring, should alter the PR-binding affinity and the fluorescence
properties.23 Therefore, in this study, we examined the structure–PR antagonist activity relationships
of coumarin derivatives bearing pyrrole (7, 9 – 11) or thiophene (8) at the 6 position (Figure 3).
Figure 3. Structures of synthesized derivatives of 6
2. Results and discussion
2.1. Synthesis of coumarin derivatives
In order to clarify the substituent effects on the 6-pyrrole ring of 6, compounds 7a – l with various
N-substituents and compounds 8a – e bearing a substituted thiophene instead of the pyrrole ring of 6
were synthesized. Then, the effects of 7-substituents, whose steric and electronic effects would

influence the properties of 6-aryl groups on the coumarin ring, were examined by synthesis of
compounds 9a – e, 10a – h, and 11 (Figure 3). In the case of compounds 10 and 11,
4-methylcoumarin derivatives were designed and synthesized for synthetic convenience.
6-Arylcoumarins 7 – 11 were prepared by Suzuki-Miyaura cross-coupling reaction of the
corresponding 6-bromocoumarin with arylboronic acid derivatives.24 Synthesis of
6-bromocoumarins 14, 18 and 21 is illustrated in Scheme 1. The Stille-Gennari modification25 of
Horner-Wadsworth-Emmons olefination of 2-acetoxy-5-bromobenzaldehyde (13), prepared by
acetylation of 12 (quant), afforded a mixture of 6-bromocoumarin and acyclic methyl cinnnamate
derivative. Treatment of the mixture with hydrochloric acid in methanol gave 6-bromocoumarin in
97% yield (two steps). 4-Diethylaminosalicylic acid was acetylated (quant), followed by
bromination (49%) to afford 17. The Stille-Gennari modification of Horner-Wadsworth-Emmons
olefination of 17 afforded 18 in 48% yield. Derivative 21 with a 6-methoxy group was prepared by
reaction of 4-bromoresorcinol with ethyl acetoacetate (75%), followed by O-methylation (quant).
Scheme 1. Synthesis of 6-bromocoumarins 14, 18 and 21. Reagents and conditions: (a) Ac2O,
pyridine, CH2Cl2; (b) (CF3CH2O)2P(O)CH2COOMe, KHMDS, 18-crown-6, THF, -78°C then rt; (c)
4 M HCl, MeOH. 85°C; (d) NBS, THF, 0°C; (e) ethyl acetoacetate, H2SO4; (f) K2CO3, MeI, DMF.
The Suzuki-Miyaura cross-coupling reactions of 14, 18, and 21 with N-Boc-pyrrole-2-boronic acid
in the presence of CsF and PdCl2(dppf) in DMF afforded 6-(N-Boc-2-pyrrolyl)coumarins 22 – 24,
respectively. Reaction of 22 – 24 with chlorosulfonyl isocyanate gave 5-cyano derivatives 25 – 27,
respectively.21 After deprotection of the Boc group by heating in water (for 25 and 27) or with
hydrochloric acid in ethanol (for 26), various N-substituents were introduced to obtain 7, 9, and 10.
6-Hydroxycoumarin derivative 11 was prepared by demethylation of 10b using boron tribromide
(44%). 6-Thiophenylcoumarins 8 were also prepared from 14 by Suzuki-Miyaura cross-coupling
reaction with substituted thiophene boronic acid or its pinacol ester.

Scheme 2. Synthesis of 6-arylcoumarins
7
-
11. Reagents and conditions: (a)
N-Boc-pyrrole-2-boronic acid, CsF, PdCl2(dppf), DMF, 60°C; (b) ClSO2NCO, THF, -78°C, then
DMF, rt; (c) H2O, 100°C (for 25 and 27); (d) 8% HCl in EtOH, 50°C (for 26); (e) NaH, alkyl
halide, DMF; (f) BBr3, CH2Cl2, -78°C then rt; (g) substituted thiophene-2-boronic acid or its pinacol
ester, CsF, PdCl2(dppf), DMF, 60°C.
2.2. PR-antagonistic activity
The PR-agonistic and antagonistic activities of the coumarin derivatives were evaluated by means
of alkaline phosphatase (AP) assay22 using T-47D human breast carcinoma cell line. None of the test
compounds alone induced alkaline phosphatase expression (data not shown), which means that they
did not act as PR agonists. PR-antagonistic activity of the synthesized compounds was examined in
the presence of 1 nM progesterone. First, the effect of the N-substituent on the pyrrole ring of
compound 6 was examined (Table 1). Removal of the N-methyl group of 6, yielding compound 7a,
decreased the activity, while replacement of the N-methyl group with a moderate-sized substituent,
such as n-hexyl (7c), cyclohexylmethyl (7d), or benzyl (7f), resulted in retention of potent
PR-antagonist activity. Introduction of a polar functional group, such as cyano (7e and 7g), nitro (7h),
or fluorine (7i – k), decreased the activity. Thus, a moderate-sized hydrophobic N-substituent on the
pyrrole ring is important for PR-antagonistic activity. Next, we examined the activity of
6-thiophenylcoumarin derivatives. All thiophene derivatives exhibited lower activity, independent of
the substituents on the thiophene ring. For example 5-cyanothiphen-2-ylcoumarin (8d) has an IC50
value of 0.14 M, which is comparable to that of compound 7a without the N-substituent, and a half
as potent as compound 6 with an N-methyl group. The result also supports the importance of the
N-substituent of 6-pyrrolylcoumarin derivatives.

Table 1. PR-antagonistic activity of 6-pyrrolylcoumarins 7
Compound
Substituent
R
T47D
IC50 (M)
0.50
5
6
0.065
0.14
0.22
0.034
0.031
15.8
0.056
0.29
0.45
0.21
0.16
0.19
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
Et
n-C6H13
CH2-c-C6H11
CH2CN
CH2Ph
CH2Ph-p-CN
CH2Ph-p-NO2
CH2Ph-p-F
CH2Ph-3,5-F2
CH2C6F5
CH2-2-naphthyl
7j
7k
7l
0.29
Table 2. PR-antagonistic activity of 6-thiophenylcoumarins 8
Compound
Substituent
T47D
IC50 (M)
0.15
R1
Me
Cl
H
R2
H
H
Me
CN
COCH3
8a
8b
8c
8d
8e
0.23
0.44
0.14
0.34
H
H
Finally, the effect of 7-substituents on the coumarin ring was examined. Besides compounds 7
lacking a 7-substituent, derivatives with a 7-diethylamino group (compounds 9), 7-methoxy group
(compounds 10) and 7-hydroxy group (compound 10) were synthesized. 7-Diethylaminocoumarin
derivatives having a rather small N-substituent on the pyrrole ring showed moderate activity, and a
larger N-substituent decreased the activity. 7-Methoxycoumarin derivatives with an N-methyl
(compound 10b) or N-ethyl (compound 10c) group showed potent PR-antagonistic activity with a
lower IC50 value (10a: 0.024 M, 10b: 0.0384 M) than that of compound 6 (IC50: 0.065 M)
lacking a 7-substituent. Introduction of a bulky N-substituent on the pyrrole ring of
7-methoxycoumarin derivatives decreased the activity, which is different from the case of
7-unsubstituted coumarin derivatives. Coumarin derivative 11 with a 7-hydroxy group showed weak
activity, compared to the 7-methoxy derivative 10b. Thus, the PR-antagonistic activity of
6-pyrrolylcoumarin depended on the hydrophobicity and bulkiness of the 7-substituent on the

coumarin ring and the N-substituent on the pyrrole ring.
Table 3. PR-antagonistic activity of 7-substituted 6-pyrrolylcoumarins 9 - 11
Compound
Substituent (R)
T47D
IC50 (M)
0.12
9a
9b
H
Me
0.24
9c
Et
0.25
9d
9e
CH2CN
CH2Ph
H
Me
Et
n-C6H13
CH2-c-C6H11
CH2Ph
CH2Ph-p-CN
CH2Ph-p-F
Me
0.85
0.58
0.26
0.024
0.038
0.18
0.55
0.23
10a
10b
10c
10d
10e
10f
10g
10h
11
0.48
0.57
0.3
The PR-binding affinity of selected compounds was evaluated by means of competitive binding
assay using the PR LBD and [3H]progesterone (Table 4). Our previous study showed that 6-aryl
derivatives with a 7-diethylamino group such as 5 had high PR-binding affinity, while
7-unsubstituted derivatives, such as 6 with higher potency in AP assay, had low PR-binding
affinity.22 In this study, we found that introduction of 7-diethylamino or 7-methoxy group into the
structure of 6 resulted in a dramatic improvement of the PR-binding affinity; the IC50 values of
compounds 9b (1.36 M) and 10b (0.99 M) are comparable to or better than that of 6 (1.42 M).
Among 7-unsubstituted derivatives, compounds 7c and 7f, which showed potent PR-antagonistic
activity in AP assay, had also higher PR-binding affinity than 6.
Table 4. PR-Binding affinity of 6-pyrrolylcoumarins
Compound
Binding
IC50a (M)
1.42
5
6
15.2
7c
7f
9b
10b
11
0.16
0.79
1.36
0.99
15.7
a[3H]Progesterone concentration was 4 nM.

Some PR ligands can bind to other steroid hormone receptors. Among our previously reported
6-arylcoumarins, compound 5 binds to androgen receptor (AR) and glucocorticoid receptor (GR),
but compound 6 does not. To examine the PR selectivity of the present compounds, we focused on
9b and 10b, which have the same 6-aryl group as 6, and examined their activity towards other
nuclear receptors. Androgenic activity was examined in terms of the effect on the growth of
androgen-dependent SC-3 cells.26 Neither 9b nor 10b alone affected the growth of SC-3 cells.
AR-antagonistic activity was evaluated in terms of the effect of test compounds on the cell growth
induced by 1 nM dihydrotestosterone (DHT). Compound 10b showed AR-antagonistic activity with
similar potency to the well-known AR antagonist, hydroxyflutamide (Figure 4), while 9b showed
AR-inhibitory activity only at 10-5 M. The GR-binding affinity of 9b and 10b was examined by
competitive binding assay with hGR and [3H]dexamethasone.22 Only compound 9b showed binding
affinity, with an IC50 value of 10.0 M, which is weaker than compound 5 (IC50: 3.54 M). Neither
of the compounds showed mineralocorticoid receptor (MR)-binding affinity in competitive binding
assay using the rMR and [3H]aldosterone (data not shown).22 Thus, the PR selectivity of 9b and 10b
was improved, although some cross-reactivity remained.
Figure 4. AR-antagonistic activity in SC-3 cell growth assay. Vertical scale is the relative cell
number when the cell number of the control (solvent only) is taken as 1. OHF: Hydroxyflutamide.
Table 5. GR- and MR-Binding affinity of 6-pyrrolylcoumarins
Compound
IC50 (M)a
hGR
3.54
ND
rMR
ND
ND
ND
ND
5
6
9b
10.0
ND
10b
aND: no detectable binding

2.3. Fluorescence properties
Compound 5 exhibited solvent-dependent fluorescence; that is, strong fluorescence was observed in
methylene chloride (quantum yield, fl: 0.744), while almost no fluorescence was seen in polar
solvents such as methanol (fl: 0.007) and 10 mM sodium phosphate buffer (pH 7.4, fl: 0.007).22
The fluorescence intensity of 5 in the buffer increased upon addition of PR LBD. Solvent
dependency of the coumarin derivative without 7-substituent is less than compound 5 with
7-diethylamino group. Therefore, we examined the flouresnt properties of 9b with 7-methoxy group
and 10b with 7-hydroxy group.
Solvent-dependent florescence was also observed with compound 9b (Figure 5a, Table 6): the
fluorescence intensity of 9b was strong in methylene chloride (fl: 0.892), weak in polar solvents
such as acetone (fl: 0.125) and acetonitrile (fl: 0.057), and almost absent in methanol (fl: 0.006)
and 10 mM sodium sodium phosphate buffer (pH 7.4, fl: 0.004). The florescence quantum yield of
9b (fl: 0.892) in methylene chloride was larger than that of 5 (fl: 0.744). Interestingly, the
fluorescence of compound 10b showed a different type of solvent-dependency: the fluorescence
intensity showed little dependence on the solvent, except in buffer, but the fluorescence maximum
wavelength shifted in the range of 419 (methylene chloride) to 466 nm (methanol). Previously, we
have reported some 6-arylcoumarin sensors in which an analyte recognition site was introduced at
the 6-aryl group. In these sensors, the mode of fluorescence change induced by binding to the
analyte was a change of fluorescence intensity for compounds with a 7-diethylamino group, and a
change of fluorescence maximum wavelength for compounds with a 7-methoxy group, which is
similar to solvent-dependency of compounds 9b and 10b.23 Although the quantum yield of 10b-type
compounds should be improved, fluorescent ligands with different modes of fluorescence change
would be useful for development of fluorescent probes for analysis of PR functions. Finally, the
fluorescence spectra of 9b in the presence of PR LBD were examined (Figure 6). Compound 9b
alone did not show fluorescence in buffer (20 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 5 mM
DTT, pH 8.0), but addition of PR LBD increased the fluorescence intensity. The increase of
florescence intensity of 9b upon addition of PR LBD (500 nM) was larger than that of 5,20 probably
due to both larger quantum yield and higher PR binding affinity of 9b, compared to those of 5. Thus,
compound 9b should be a more sensitive sensor for PR, compared to previously reported 5.

(a)
(b)
CH2Cl2
(CH3)2CO
CH3CN
CH3OH
Buffer
CH2Cl2
(CH3)2CO
CH3CN
CH3OH
Buffer
Figure 5. Fluorescence spectra of (a) 9b and (b) 10b (2 M) in various solvents. Buffer is 10 mM
sodium phosphate buffer (pH 7.4). Each solvent contained 0.2% DMSO as a cosolvent. Excitation
wavelengths were 350 nm for 9b and 330 nm for 10b.
Table 6. Fluorescence properties of coumarin derivatives 9b and 10b
Compound
Solventa
CH2Cl2
452
(CH3) 2CO
464
CH3CN
CH3OH
460
bufferb
470
Emmax, nm
467
9b
c
0.892
419
0.125
430
0.057
437
0.006
466
0.004
486
fl
Emmax, nm
10b
c
0.139
0.056
0.076
0.113
0.015
fl
aEach solvent contained 0.2% DMSO as a cosolvent.
bBuffer is 10 mM sodium phosphate buffer (pH 7.4).
cFluorescence quantum yields (fl) were determined using quinine sulfate (0.577) in 0.1 M sulfuric
acid as a standard.
Figure 6. Fluorescence spectra of 9b (1 M) in the presence of PR LBD in buffer (20 mM
Tris-HCl, 300 mM NaCl, 1 mM EDTA, 5 mM DTT, pH 8.0). Excitation wavelength was 360 nm.
3. Conclusion
We designed and synthesized novel coumarins bearing an aromatic heterocycle as nonsteroidal PR
antagonists by using 5 and 6 as lead compounds. Some compounds, such as 7c and 10b, showed

more potent PR-antagonistic activity than the lead compounds in AP assay and PR-binding
experiments. None of them showed PR-agonistic activity in AP assay, and therefore 6-arylcoumarin
should be a useful scaffold for development of PR antagonists, since some of the known PR ligands
can act as either agonists or antagonists depending on their substituents. There were two modes of
fluorescence change in various solvents, that is, change of fluorescence intensity (9b) and change of
fluorescence maximum wavelength (10b). Compound 9b showed no fluorescence in buffer alone,
but showed strong fluorescence in the presence of PR LBD, and therefore is a sensor candidate for
PR functions. The change of fluorescence maximum wavelength exhibited by 10b would be a useful
property for a PR sensor, since the compound could be employed for ratiometric measurement,
although the quantum yield would need to be improved. Our findings here should contribute to the
development of novel nonsteroidal PR ligands and sensors.
4. Experimental
4.1. Chemistry
All reagents were purchased from Sigma-Aldrich Chemical Co., Tokyo Kasei Kogyo Co., Wako
Pure Chemical Industries, and Kanto Kagaku Co., Inc. Silica gel for column chromatography was
purchased from Kanto Kagaku Co., Inc. 1H and 13C NMR spectra were recorded on a JEOL ECS 400
spectrometer or a Bruker 600 spectrometer. Mass spectral data was obtained on a Bruker Daltonics
microTOF-2focus or MStation JMS-700 in the positive ion detection mode.
4.2. Synthesis
4.2.1. Synthesis of 2-acetoxy-5-bromobenzaldehyde (13)
Acetic anhydride (11 mL) was added to a suspension of 12 (5.01 g, 24.9 mmol) in dry pyridine (20
mL) and dry methylene chloride (60 mL) at room temperature. After 1 h, the solvents were removed
in vacuo to afford 13 (5.76 g, 95%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 10.1 (1 H, s),
8.00 (1 H, d, J = 2.8 Hz), 7.73 (1 H, dd, J = 2.3, 8.7 Hz), 7.10 (1 H, d, J = 8.7 Hz), 2.39 (3 H, s); 13C
NMR (150MHz, CDCl3) δ 187.3, 169.0, 150.7, 138.1, 133.6, 129.4, 125.5, 119.9, 20.9.
4.2.2. Synthesis of 5-bromocoumarin (14)
Potassium bis(trimethylsilyl)amide (0.5 M solution in toluene, 34 mL, 17.0 mmol) was added to a
solution of bis(2,2,2-trifluoromethyl)(methoxycarbonylmethyl)phosphonate (5.58 g, 17.6 mmol) and
18-crown-6 (7.59 g, 28.7 mmol) in dry THF (100 mL) at 0°C under argon. Compound 13 (3.87 g,
15.9 mmol) was added at -78°C. The reaction mixture was stirred at room temperature for 3 h,
neutralized with ammonium chloride, and then extracted with ethyl acetate. The organic layer was
dried over magnesium sulfate, filtered, and concentrated. Methanol (10 mL) and 4 M hydrochloric
acid (6 mL) were added to the residue, and the mixture was refluxed at 85°C for 30 min, neutralized

with sodium bicarbonate, and extracted with ethyl acetate. The organic layer was washed with brine,
dried over magnesium sulfate, filtered, and concentrated. The residue was purified by column
1
chromatography (silica gel, AcOEt / n-hexane = 1/4) to give 32 (3.47 g, 97%). H NMR (400 MHz,
CDCl3) δ 7.65-7.61 (3 H, m), 7.23 (1 H, dd, J = 2.8, 8.0 Hz), 6.46 (1 H, d, J = 9.6 Hz); 13C NMR
(150 MHz, CDCl3) δ 160.0, 153.0, 142.2, 134.7, 130.2, 120.4, 118.7, 118.0, 117.1.
4.2.3. Synthesis of 2-acetoxy-4-(diethylamino)benzaldehyde (16)
Acetic anhydride (7.0 mL) was added to a suspension of 15 (2.54 g, 13.2 mmol) in dry pyridine (10
mL) and dry methylene chloride (30 mL) at room temperature. After 30 min, the solvents were
1
removed in vacuo to afford 16 (3.30 g, quant.) as a pale yellow oil. H NMR (400 MHz, CDCl3) δ
9.74 (1 H, s), 7.65 (1 H, d, J = 8.8 Hz), 6.55 (1 H, dd, J = 2.4, 8.8 Hz), 6.26 (1 H, d, J = 2.4 Hz), 3.41
(4 H, q, J = 7.2 Hz), 2.38 (3 H, s), 1.21 (6 H, t, J = 6.8 Hz).
4.2.4. Synthesis of 2-acetoxy-5-bromo-4-(diethylamino)benzaldehyde (17)
Bromine (2.87 g, 18.0 mmol) in acetic acid (15 mL) was added to a suspension of 16 (3.17 g, 13.5
mmol) in acetic acid (5 mL) at 0°C. The reaction mixture was stirred at room temperature for 4 h.
The reaction was quenched with water at 0°C, and the mixture was neutralized with sodium
bicarbonate, and extracted with ethyl acetate. The organic layer was washed with brine, dried over
magnesium sulfate, filtered, and concentrated. The residue was purified by column chromatography
1
(silica gel, AcOEt / n-hexane = 1/5) to give 16 (2.05 g, 48%) as a brown oil. H NMR (400 MHz,
CDCl3) δ 9.86 (1 H, s), 8.00 (1 H, s), 6.70 (1 H, s), 3.28 (4 H, q, J = 7.6 Hz), 2.38 (3 H, s), 1.14 (6 H,
t, J = 6.9 Hz) ; 13C NMR (150 MHz, CDCl3) δ 163.4, 153.8, 111.2, 109.6, 104.5, 99.5, 96.2, 95.9,
44.2, 12.5, 12.2.
4.2.5. Synthesis of 5-bromo-6-(diethylamino)coumarin (18)
Potassium bis(trimethylsylyl)amide (0.5 M solution in toluene, 17 mL, 8.50 mmol) was added to a
solution of bis(2,2,2-trifluoromethyl)(methoxycarbonylmethyl)phosphonate (0.90 mL) and
18-crown-6 (2.96 g, 11.2 mmol) in dry THF (66 mL) at 0°C under argon. Compound 17 (1.32 g,
4.21 mmol) was added to the mixture at −78°C. The reaction mixture was stirred at room
temperature for 1 d, and then neutralized with ammonium chloride, and extracted with ethyl acetate.
The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was
purified by column chromatography (silica gel, AcOEt / n-hexane = 1/5) to give 18 (601 mg, 48%)
1
as a yellow oil. H NMR (400 MHz, CDCl3) δ 7.66 (1 H, s), 7.56 (1 H, d, J = 8.8 Hz), 6.94 (1 H, s),
6.28 (1 H, d, J = 9.6 Hz), 3.22 (4 H, q, J = 7.2 Hz), 1.10 (6 H, t, J = 7.2 Hz); 13C NMR (150 MHz,
CDCl3) δ 160.9, 154.1, 152.9, 142.3, 132.4, 115.2, 114.7, 114.6, 110.6, 46.4, 12.2.

4.2.6. Synthesis of 5-bromo-6-hydroxy-4-methylcoumarin (20)
A mixture of 4-bromoresorcinol (19, 2.29 g, 12.1 mmol), 3-oxobutanoic acid (1.6 mL), and conc.
H2SO4 (15 mL) was stirred at 0°C to room temperature for 1 d. The reaction mixture was poured into
ice water, and the precipitates were collected and washed with water to give 20 (2.23 g, 75%). 1H
NMR (400 MHz, CDCl3) δ 7.70 (1 H, s), 7.00 (1 H, s), 6.18 (1 H, s), 2.39 (3 H, s).
4.2.7. Synthesis of 5-bromo-6-methoxy-4-methylcoumarin (21)
A mixture of 41 (763 mg, 2.99 mmol), K2CO3 (516 mg, 3.73 mmol) in DMF (8.0 mL) was stirred for
15 min. Iodoethane (0.25 mL, 4.02 mmol) was added, and the reaction mixture was stirred for 1 d at
room temperature. The mixture was quenched with 2 M hydrochloric acid, and extracted with ethyl
acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered, and
concentrated to give 21 (809 mg, quant). 1H NMR (400 MHz, CDCl3) δ 7.75 (1 H, s), 6.84 (1 H, s),
6.17 (1 H, s), 3.96 (3 H, s), 2.39 (3 H, s); 13C NMR (150 MHz, CDCl3) δ 160.8, 158.5, 154.4, 151.7,
128.7, 114.6, 112.9, 107.5, 100.4, 56.9, 18.8.
4.2.8. Typical procedure for compounds 22
–
24 and 8a
–
f: Synthesis of
5-(N-tert-butoxycarbonyl-2-pyrrolyl)-6-(diethylamino)coumarin (23)
18 (138.5 mg, 0.47 mmol) was added to a solution of boronic acid (304.5 mg, 1.44 mmol), cesium
fluoride (303 mg, 1.99 mmol), and PdCl2(dppf) (81.2 mg, 0.10 mmol) in dry DMF (5 mL) at room
temperature under argon. The reaction mixture was heated at 60°C for 3 h, then neutralized with
ammonium chloride, and extracted with ethyl acetate. The organic layer was washed with brine,
dried over magnesium sulfate, filtered, and concentrated. The residue was purified by column
chromatography (silica gel, AcOEt / n-hexane = 1/4) to give 23 (129 mg, 72%). 1H NMR (400 MHz,
CDCl3) δ 7.58 (1 H, d, J = 9.2 Hz), 7.32 (1 H, m), 7.24 (1 H, s), 6.82 (1 H, s), 6.25 (1 H, t, J = 3.2
Hz), 6.19 (1 H, d, J = 9.2 Hz), 6.18 (1 H, m), 2.97 (4 H, br), 1.36 (9 H, s), 0.95 (6 H, t, J = 6.8 Hz);
13C NMR (150 MHz, CDCl3) δ 161.9, 155.2, 154.4, 148.9, 143.5, 132.3, 130.5, 123.9, 122.1, 113.9,
112.3, 111.5, 111.0, 106.2, 83.5, 44.3, 27.8, 12.2.
Compounds 22 and 24 were synthesized similarly from compounds 14 and 21, respectively.
1
22: 71%; H NMR (400 MHz, CDCl3) δ 7.71 (1 H, d, J = 9.6 Hz), 7.52 (1 H, dd, J = 2.3, 8.7 Hz),
7.47 (1 H, d, J = 2.3 Hz), 7.36 (1 H, m), 7.32 (1 H, d, J = 8.3 Hz), 6.45 (1 H, d, J = 9.2 Hz), 6.25 (1
H, t, J = 3.2 Hz), 6.22 (1 H, m), 1.40 (9 H, s); 13C NMR (150 MHz, CDCl3) δ 160.8, 153.2, 149.1,
143.4, 133.2, 133.0, 130.9, 128.0, 122.9, 118.1, 116.9, 116.1, 115.1, 110.8, 84.0, 27.2.
24: 91%; 1H NMR (400 MHz, CDCl3) δ 7.47 (1 H, s), 7.37 (1 H, m), 6.81 (1 H, s), 6.27(1 H, t, J =
3.2 Hz), 6.19 (1 H, m), 6.16 (1 H, d, J = 1.4 Hz), 3.83 (3 H, s), 2.40 (3 H, d, J = 0.9 Hz), 1.40 (9 H,
s)

Compounds 8a - e were synthesized similarly from compounds 14.
1
8a: 99%; H NMR (400 MHz, CDCl3) δ 7.74 (1 H, d, J = 9.6 Hz), 7.62 (1 H, dd, J = 2.3, 8.7 Hz),
7.54 (1 H, d, J = 2.3 Hz), 7.38 (1 H, d, J = 8.7 Hz), 7.25 (1 H, d, J = 5.0 Hz), 6.95 (1 H, d, J = 5.0
Hz), 6.47 (1 H, d, J = 9.6 Hz), 2.32 (3 H, s); 13C NMR (150 MHz, CDCl3) δ 160.8, 153.2, 143.4,
135.8, 134.0, 132.7, 131.5, 131.3, 128.0, 124.1, 119.0, 117.3, 117.2, 15.0; HRMS (ESI+) Calcd. for
C14H11O2S [M + H]+ : 243.0474. Found 243.0470.
1
8b: 44%; H NMR (400 MHz, CDCl3) δ 7.81 (1 H, dd, J = 2.0, 8.4 Hz), 7.78 (1 H, d, J = 2.0 Hz),
7.75 (1 H, d, J = 9.6 Hz), 7.39 (1 H, d, J = 8.8 Hz), 7.31 (1 H, d, J = 5.6 Hz), 7.02 (1 H, d, J = 5.6
Hz), 6.48 (1 H, d, J = 9.6 Hz); 13C NMR (150 MHz, CDCl3) δ 160.3, 153.6, 143.5, 134.2, 132.4,
130.4, 129.6, 128.8, 127.8, 124.4, 118.9, 118.0, 117.4; HRMS (ESI+) Calcd. for C13H7ClNaO2S [M
+ Na]+ : 284.9747. Found 284.9751.
1
8c: 23%; H NMR (400 MHz, CDCl3) δ7.72 (1 H, d, J = 9.6 Hz), 7.70 (1 H, dd, J = 1.8, 8.5 Hz),
7.60 (1 H, d, J = 1.8 Hz), 7.32 (1 H, d, J = 8.7 Hz), 7.11 (1 H, d, J = 3.7 Hz), 6.75 (1 H, m), 6.45 (1
H, d, J = 9.6 Hz), 2.52 (3 H, s); 13C NMR (150 MHz, CDCl3) δ 160.8, 153.1, 143.5, 140.5, 139.9,
131.6, 129.2, 126.6, 124.2, 123.6, 119.2, 117.5, 117.3, 15.6; HRMS (ESI+) Calcd. for C14H11NaO2S
[M + Na]+ : 265.0294. Found 265.0293.
1
8d: 27%; H NMR (400 MHz, CDCl3) δ 7.75 (1 H, d, J = 9.6 Hz), 7.75 (1 H, dd, J = 2.3, 8.0 Hz),
7.69 (1 H, d, J = 1.8 Hz), 7.62 (1 H, d, J = 3.7 Hz), 7.41 (1 H, d, J = 8.7 Hz), 7.29 (1 H, d, J = 4.1
Hz), 6.51 (1 H, d, J = 9.6 Hz); 13C NMR (150 MHz, CDCl3) δ 160.1, 154.6, 149.7, 142.9, 138.7,
129.9, 129.0, 125.6, 124.0, 119.5, 118.2, 118.1, 114.2, 109.1; HRMS (ESI+) Calcd. for C14H8NO2S
[M + H]+ : 254.0270. Found 254.0274.
1
8e: 33%; H NMR (400 MHz, CDCl3) δ 7.80 (1 H, dd, J = 2.3, 8.5 Hz), 7.74 (1 H, d, J = 9.6Hz),
7.74 (1 H, d, J = 2.3 Hz), 7.68 (1 H, d, J = 3.7 Hz), 7.39 (1 H, d, J = 8.2 Hz), 7.33 (1 H, d, J = 4.1
Hz), 6.50 (1 H, d, J = 9.6 Hz), 2.58 (3 H, s); 13C NMR (150 MHz, CDCl3) δ 190.4, 160.1, 154.3,
150.4, 143.8, 142.9, 133.4, 130.0, 129.5, 125.3, 124.3, 119.3, 117.8, 26.6; HRMS (ESI+) Calcd. for
C15H11O3S [M + H]+ : 271.0423. Found 271.0426.
4.2.9.
Typical
procedure
for
compounds
25
–
27:
Synthesis
of
5-(N-tert-butoxycarbonyl-5-cyano-2-pyrrolyl)-6-(diethylamino)coumarin (26)
Chlorosulfonyl isocyanate (0.3 mL) was added to a solution of 23 (376 mg, 0.98 mmol) in dry THF
(20 mL) at -78°C. After 1 h, DMF (0.5 mL) was added. The reaction mixture was allowed to warm

to room temperature, then poured into water, and extracted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by
1
column chromatography (silica gel, AcOEt / n-hexane = 1/3) to give 26 (309 mg, 77%). H NMR
(400 MHz, CDCl3) δ7.57 (1 H, d, J = 9.2 Hz), 7.25 (1 H, s), 7.02 (1 H, d, J = 3.6 Hz), 6.87 (1 H, s),
6.27 (1 H, d, J = 3.6 Hz), 6.24 (1 H, d, J = 9.2 Hz), 2.95 (4 H, br), 1.49 (9 H, s), 0.97 (6 H, t, J = 6.8
Hz); 13C NMR (150 MHz, CDCl3) δ 161.3, 155.6, 154.1, 146.8, 143.1, 138.1, 130.2, 124.7, 122.3,
113.5, 113.3, 112.2, 107.2, 105.1, 87.1, 44.5, 27.7, 11.9.
Compounds 25 and 27 were synthesized similarly from compounds 22 and 24, respectively.
1
25: 90%; H NMR (400 MHz, CDCl3) δ 7.71 (1 H, d, J = 9.6 Hz), 7.49 (1 H, dd, J = 1.8, 8.6 Hz),
7.48 (1 H, d, J = 1.8 Hz), 7.37 (1 H, d, J = 8.7 Hz), 7.01 (1 H, m), 6.48 (1 H, d, J = 9.6 Hz), 6.29 (1
H,m), 1.50 (9H, s); 13C NMR (150 MHz, CDCl3) δ 160.5, 154.0, 147.2, 143.1, 138.5, 132.8, 128.9,
128.2, 124.6, 118.5, 117.6, 116.8, 114.7, 113.3, 106.1, 87.7, 27.7.
27: 1H NMR (400 MHz, CDCl3) δ 7.47 (1 H, s), 7.00 (1 H, d, J = 3.7 Hz), 6.84 (1 H, s), 6.24 (1 H, d,
J = 3.7 Hz), 6.19 (1 H, d, J = 0.9 Hz), 3.84 (3 H, s), 2.41 (3 H, d, J = 0.9 Hz), 1.51 (9 H, s).
4.2.10.
Typical
procedure
for
compounds
7a
and
10a:
Synthesis
of
5-(5-cyano-2-pyrrolyl)coumarin (7a)
A solution of 25 (888 mg, 2.64 mmol) in water (50 mL) was heated at 100°C for 3 h. The reaction
mixture was extracted with AcOEt. The organic layer was washed with brine, dried over sodium
1
sulfate, filtered, and concentrated to give 7a (621mg, quant.). H NMR (400 MHz, CDCl3) δ9.26
(1 H, br), 7.74 (1 H, d, J = 9.6 Hz), 7.68 (1 H, dd, J = 2.4, 8.6 Hz), 7.62 (1 H, d, J = 2.4 Hz), 7.40 (1
H, d, J = 8.8 Hz), 6.94 (1 H, m), 6.55 (1 H, m), 6.50 (1 H, d, J = 9.6 Hz); 13C NMR (150 MHz,
DMSO-d6) δ 159.7, 152.8, 144.0, 135.6, 128.3, 127.3, 124.3, 121.0, 119.1, 117.0, 116.9, 114.8, 107.7,
100.9; HRMS (ESI+) Calcd. for C14H9N2O2 [M + H]+ : 237.0659. Found 237.0658.
1
Compounds 10a was synthesized similarly from compound 27. H NMR (400 MHz, CDCl3) δ 7.81
(1 H, s), 6.96 (1 H, s), 6.92 (1 H, m), 6.63 (1 H, m), 6.22 (1 H, d, J = 1.2 Hz), 4.10 (3 H, s), 2.46 (3
H, d, J = 1.2 Hz); 13C NMR (150 MHz, CDCl3) δ 160.0, 158.8, 154.0, 153.6, 132.6, 123.9, 120.4,
116.6, 115.2, 113.1, 111.8, 110.7, 100.2, 100.1, 56.6, 18.3; HRMS (ESI+) Calcd. for C16H13N2O3 [M
+ H]+ : 281.0921. Found 281.0922.
4.2.11. Synthesis of 5-(5-cyano-2-pyrrolyl)-6-(diethylamino)coumarin (9a)
A solution of 26 (61.9 mg, 0.20 mmol) in 8% hydrochloric acid/ethanol (3 mL) was heated at 60°C
for 3 h. The reaction mixture was extracted with methylene chloride. The organic layer was washed
with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column

1
chromatography (silica gel, ether) to give 9a (58.7 mg, 95%) as a yellow oil. H NMR (400 MHz,
CDCl3) δ12.0 (1 H, br), 7.70 (1 H, s), 7.69 (1 H, d, J = 10.1 Hz), 7.19 (1 H, s), 6.89 (1 H, d, J = 2.3
Hz), 6.55 (1 H, d, J = 2.8 Hz), 6.40 (1 H, d, J = 9.6 Hz), 3.10 (4 H, q, J = 6.9 Hz), 1.07 (6 H, t, J =
6.9 Hz); 13C NMR (150 MHz, CDCl3) δ 160.5, 153.8, 150.8, 142.9, 135.4, 127.0, 123.8, 120.7, 116.3,
116.2, 114.9, 111.9, 107.8, 100.2, 48.4, 12.0; HRMS (ESI+) Calcd. for C18H18N3O2 [M + H]+ :
308.1394. Found 308.1392.
4.2.12. Typical procedure for N-alkylation of pyrrole ring: Synthesis of
5-(5-cyano-1-methyl-2-pyrrolyl)-6-(diethylamino)coumarin (9b)
Sodium hydride (25.2 mg, 0.63 mmol) was washed with n-hexane. A solution of 9a (46.1 mg, 0.15
mmol) in dry DMF (1.5 mL) was added to a suspension of sodium hydride in dry DMF (1 mL) at
room temperature. After 30 min, iodoethane (65.4 mg, 0.46 mmol) was added. The reaction mixture
was stirred for 2 h at room temperature, then poured into water, and extracted with ethyl acetate. The
organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated to give 9b
(44.1 mg, 92%). 1H NMR (400 MHz, CDCl3) δ 7.58 (1 H, d, J = 9.2 Hz), 7.26 (1 H, s), 6.89 (1 H, s),
6.88 (1 H, d, J = 4.0 Hz), 6.25 (1 H, d, J = 9.2 Hz), 6.18 (1 H, d, J = 3.6 Hz), 3.52 (3 H, s), 3.00 (4 H,
q, J = 7.6 Hz), 0.95 (6 H, t, J = 7.2 Hz); 13C NMR (150 MHz, CDCl3) δ 161.0, 155.7, 153.4, 142.7,
138.3, 132.5, 120.4, 119.8, 114.1, 113.3, 112.0, 109.6, 106.6, 104.7, 45.6, 33.4, 12.2; HRMS (ESI+)
Calcd. for C19H20N3O2 [M + H]+ : 322.1550. Found 320.1556.
Compounds 7b-l, 9c-e, and 10b-h were synthesized similarly.
7b: 95%; 1H NMR (400 MHz, CDCl3) δ7.73 (1 H, d, J = 9.6 Hz), 7.53 (1 H, dd, J = 2.4, 8.8 Hz),
7.49 (1 H, d, J = 2.0 Hz), 7.43 (1 H, d, J = 8.8 Hz,), 6.88 (1 H, d, J = 3.6 Hz), 6.51 (1 H, d, J = 9.6
Hz), 6.21 (1 H, d, J = 4.0 Hz), 4.11 (2 H, q, J = 7.2 Hz), 1.35 (3 H, t, J = 7.6 Hz); 13C NMR (150
MHz, CDCl3) δ 160.1, 154.0, 142.8, 137.4, 132.3, 128.3, 128.0, 120.0, 119.1, 117.7, 117.5, 113.9,
110.5, 104.8, 41.5, 16.7; HRMS (ESI+) Calcd. for C16H13N2O2 [M + H]+ : 265.0972. Found
265.0976.
7c: 92%; 1H NMR (400 MHz, CDCl3) δ7.72 (1 H, d, J = 9.6 Hz), 7.52 (1 H, dd, J = 2.4, 8.0 Hz),
7.48 (1 H, d, J = 2.0 Hz), 7.42 (1 H, d, J = 8.8 Hz), 6.87 (1 H, d, J = 4.0 Hz), 6.51 (1 H, d, J = 9.6
Hz), 6.21 (1 H, d, J = 4.4 Hz), 4.06 (t, J = 7.6 Hz, 2H), 1.66 (2 H, quint, J = 7.2 Hz), 1.23-1.13 (6 H,
m), 0.81 (3 H, t, J = 6.8 Hz); 13C NMR (150 MHz, CDCl3) δ 160.1, 153.9, 142.8, 137.6, 132.0, 128.3,
119.9, 119.1, 117.7, 117.4, 114.0, 110.6, 105.1, 46.6, 31.1, 31.0, 26.0, 22.3, 13.8; HRMS (ESI+)
Calcd. for C20H21N2O2 [M + H]+ : 321.1598. Found 321.1602.
7d: 99%; 1H NMR (400 MHz, CDCl3) δ 7.74 (1 H, d, J = 9.6 Hz), 7.51 (1 H, dd, J = 2.4, 8.0 Hz),

7.47 (1 H, d, J = 1.6 Hz), 7.42 (1 H, d, J = 8.8 Hz), 6.86 (1 H, d, J = 4.0 Hz), 6.51 (1 H, d, J = 9.6
Hz), 6.20 (1 H, d, J = 4.0 Hz), 3.94 (2 H, d, J = 8.0 Hz), 1.63-1.50 (4 H, m), 1.38-1.30 (2 H, m),
1.13-1.00 (3 H, m), 0.78-0.68 (2 H, m); 13C NMR (150 MHz, CDCl3) δ 160.2, 153.8, 142.9, 138.1,
132.5, 128.4, 119.8, 119.0, 117.7, 117.4, 114.2, 110.7, 105.7, 52.7, 39.3, 30.2, 25.9, 25.4; HRMS
(ESI+) Calcd. for C21H21N2O2 [M + H]+ : 333.1598. Found 333.1593.
7e: 12%; 1H NMR (400 MHz, CDCl3) δ 7.76 (1 H, d, J = 9.6 Hz), 7.59 (1 H, s), 7.58 (1 H, dd, J =
2.4, 5.6 Hz), 7.49 (1 H, d, J = 9.2 Hz), 7.00 (1 H, d, J = 4.0 Hz), 6.55 (1 H, d, J = 9.6 Hz), 6.37 (1 H,
d, J = 4.0 Hz), 4.89 (2 H, s); 13C NMR (150 MHz, DMSO-d6) δ 153.7, 143.9, 138.6, 132.2, 129.0,
125.7, 122.0, 120.5, 119.2, 117.2, 115.3, 112.8, 111.6, 105.0, 90.7, 34.4; HRMS (ESI+) Calcd. for
C16H10N3O2 [M + H]+ : 276.0768. Found 276.0767.
7f: 45%; 1H NMR (400MHz, CDCl3) δ 7.56 (1 H, d, J = 9.6 Hz), 7.42 (1 H, dd, J = 2.0, 8.8 Hz),
7.32(1 H, d, J = 2.0 Hz), 7.32 (1 H, d, J = 8.8 Hz), 7.29 (3 H, m), 6.95 (1 H, d, J = 4.0 Hz), 6.92 (2 H,
m), 6.45 (1 H, d, J = 9.6 Hz), 6.32 (1 H, d, J = 4.0 Hz), 5.27 (2 H, s); 13C NMR (150 MHz, CDCl3) δ
160.1, 154.0, 142.8, 138.3, 136.5, 132.4, 129.0, 128.4, 128.1, 127.7, 126.1, 120.3, 118.9, 117.6,
117.4, 113.9, 110.9, 106.1, 49.9; HRMS (ESI+) Calcd. for C21H15N2O2 [M + H]+ : 327.1128. Found
327.1127.
7g: 44%; 1H NMR (400 MHz, CDCl3) δ 7.61 (2 H, d, J = 8.8 Hz), 7.60 (1 H, d, J = 9.6 Hz), 7.39 (1
H, dd, J = 2.0, 8.4 Hz), 7.37 (1 H, d, J = 8.8 Hz), 7.34 (1 H, d, J = 2.0 Hz), 7.01 (2 H, d, J = 8.0 Hz),
7.00 (1 H, d, J = 4.0 Hz), 6.48 (1 H, d, J = 9.6 Hz), 6.36 (1 H, d, J = 4.0 Hz), 5.33 (2 H, s); 13C NMR
(150 MHz, CDCl3) δ 159.8, 154.1, 142.5, 141.5, 138.4, 132.8, 132.1, 128.3, 127.2, 126.7, 120.8,
119.1, 117.9, 117.6, 113.4, 112.2, 111.4, 106.0, 49.4; HRMS (ESI+) Calcd. for C22H14N3O2 [M +
H]+ : 352.1081. Found 352.1079.
7h: 59%; 1H NMR (400 MHz, CDCl3) δ8.17 (2 H, d, J = 8.8 HzH), 7.61 (1 H, d, J = 9.6 Hz), 7.40 (1
H, dd, J = 2.0, 8.4 Hz), 7.37 (1 H, d, J = 2.0 Hz), 7.35 (1 H, d, J = 8.4 Hz), 7.07 (2 H, d, J = 9.2 Hz),
7.01 (1 H, d, J = 4.4 Hz), 6.48 (1 H, d, J = 9.6 Hz), 6.38 (1 H, d, J = 3.6 Hz), 5.38 (2 H, s); 13C NMR
(150 MHz, CDCl3) δ 159.9, 154.1, 147.6, 143.5, 142.5, 138.4, 132.1, 128.3, 127.4, 126.9, 124.3,
120.9, 119.2, 118.0, 117.7, 113.3, 111.5, 106.0, 49.2; HRMS (ESI+) Calcd. for C21H14N3O4 [M +
H]+ : 372.0979. Found 372.0976.
7i: 49%; 1H NMR (400 MHz, CDCl3) δ 7.60 (1 H, d, J = 9.6 Hz), 7.42 (1 H, dd, J = 2.0, 8.8 Hz),
7.35 (1 H, d, J = 8.4 Hz), 7.34 (1 H, d, J = 2.0 Hz), 6.99 (2 H, t, J = 8.8 Hz), 6.96 (1 H, d, J = 4.0
Hz), 6.89 (2 H, dd, J = 5.6, 8.8 Hz), 6.48 (1 H, d, J = 9.6 Hz), 6.31 (1 H, d, J = 4.0 Hz), 5.24 (2 H, s);

13C NMR (150 MHz, CDCl3) δ 163.5, 161.1, 160.0, 154.0, 142.7, 138.2, 132.2, 128.4, 128.0, 120.4,
119.0, 117.7, 117.4, 116.1, 115.9, 113.7, 111.1, 105.9, 49.2; HRMS (ESI+) Calcd. for C21H14FN2O2
[M + H]+ : 345.1034. Found 345.1039.
7j: 37%; 1H NMR (400 MHz, CDCl3) δ 7.62 (1 H, d, J = 9.6 Hz), 7.41 (1 H, dd, J = 1.6, 8.4 Hz),
7.37 (1 H, d, J = 1.6 Hz), 7.36 (1 H, d, J = 8.0 Hz), 7.00 (1 H, d, J = 4.4 Hz), 6.74 (1 H, tt, J = 2.4,
8.8 Hz), 6.48 (1 H, d, J = 9.6 Hz), 6.43 (2 H, br d, J = 6.0 Hz), 6.36 (1 H, d, J = 4.0 Hz), 5.25 (2 H,
s); 13C NMR (150 MHz, CDCl3) δ 164.6, 162.1, 159.9, 154.1, 142.6, 140.4, 138.3, 132.2, 128.3,
127.2, 120.8, 119.1, 117.9, 117.6, 113.4, 111.3, 109.1, 106.0, 103.8, 49.0; HRMS (ESI+) Calcd. for
C21H12F2N2NaO2 [M + Na]+ : 385.0759. Found 385.0760.
7k: 51%; 1H NMR (400 MHz, CDCl3) δ 7.71 (1 H, d, J = 9.6 Hz), 7.46 (1 H, dd, J = 1.6, 6.4 Hz),
7.45 (1 H, d, J = 1.6 Hz), 7.41 (1 H, d, J = 9.2 Hz), 6.93 (1 H, d, J = 4.4 Hz), 6.52 (1 H, d, J = 9.6
Hz), 6.25 (1 H, d, J = 4.0 Hz), 5.35 (2 H, s); 13C NMR (150 MHz, CDCl3) δ 159.9, 154.2, 142.5,
138.6, 132.6, 128.7, 127.2, 121.1, 119.1, 118.0, 117.6, 117.6, 113.0, 111.2, 105.7, 38.6; HRMS
(ESI+) Calcd. for C21H10F5N2O2 [M + H]+ : 417.0657. Found 417.0650.
7l: 27%; 1H NMR (400 MHz, CDCl3) δ 7.83-7.79 (2 H, m), 7.71-7.69 (1 H, m), 7.56-7.47 (3 H, m),
7.44 (1 H, dd, J = 2.4, 8.8 Hz), 7.31-7.26 (3 H, m), 7.09 (1 H, dd, J = 2.0, 8.0 Hz), 7.00 (1 H, d, J =
4.0 Hz), 6.41 (1 H, d, J = 9.6 Hz), 6.35 (1 H, d, J = 4.0 Hz), 5.34 (2 H, s); 13C NMR (150 MHz,
CDCl3) δ 160.1, 153.9, 142.7, 138.3, 134.0, 133.2, 132.8, 132.4, 129.0, 128.4, 127.8, 127.7, 127.7,
126.6, 126.4, 125.1, 123.7, 120.3, 118.9, 117.5, 117.3, 113.8, 111.0, 106.1, 50.1; HRMS (ESI+)
Calcd. for C25H17N2O2 [M + H]+ : 377.1285. Found 377.1274.
9c: 99%; 1H NMR (400 MHz, CDCl3) δ 7.58 (1 H, d, J = 9.2 Hz), 7.26 (1 H, s), 6.90 (1 H, d, J = 4.0
Hz), 6.88 (1 H, s), 6.25 (1 H, d, J = 8.8 Hz), 6.14 (1 H, d, J = 4.0 Hz), 3.95 (2 H, q, J = 7.2 Hz), 3.01
(4 H, q, J = 7.2 Hz), 1.17 (3 H, t, J = 7.6 Hz), 0.95 (6 H, t, J = 6.8 Hz); 13C NMR (150 MHz, CDCl3)
δ 160.1, 154.0, 142.8, 137.4, 132.3, 128.3, 128.0, 120.0, 119.0, 117.7, 117.5, 113.9, 110.6, 104.8,
41.5, 16.7; HRMS (ESI+) Calcd. for C20H22N3O2 [M + H]+ : 336.1707. Found 336.1706.
9d: 37%; 1H NMR (400 MHz, CDCl3) δ 7.60 (1 H, d, J = 9.2 Hz), 7.35 (1 H, s), 7.01 (1 H, d, J = 4.0
Hz), 6.97 (1 H, s), 6.30 (1 H, d, J = 9.2 Hz), 6.30 (1 H, d, J = 4.0 Hz), 4.54 (2 H, s), 3.03 (4 H, q, J =
6.8 Hz), 0.99 (6 H, t, J = 6.8 Hz); 13C NMR (150 MHz, CDCl3) δ 160.6, 156.1, 152.5, 142.6, 138.5,
133.0, 122.0, 119.0, 114.4, 113.0, 112.4, 111.9, 111.1, 108.0, 105.2, 46.2, 34.3, 12.0; HRMS (ESI+)
Calcd. for C20H19N4O2 [M + H]+ : 347.1503. Found 347.1494.

9e: 36%; 1H NMR (400 MHz, CDCl3) δ 7.42 (1 H, d, J = 9.2 Hz), 7.14 (3 H, m), 7.02 (1 H, s), 6.94
(1 H, d, J = 4.0 Hz), 6.89 (1 H, s), 6.71 (2 H, m), 6.20 (1 H, d, J = 9.6 Hz), 6.20 (1 H, d, J = 3.6 Hz),
5.12 (2 H, s), 3.02 (4 H, q, J = 7.2 Hz), 0.99 (6 H, t, J = 6.8 Hz); 13C NMR (150 MHz, CDCl3) δ
161.2, 155.7, 153.2, 142.9, 138.2, 136.5, 133.1, 128.7, 127.9, 127.1, 120.9, 120.7, 114.4, 113.3,
112.13, 110.4, 106.7, 104.8, 50.6, 45.9, 12.4; HRMS (ESI+) Calcd. for C25H24N3O2 [M + H]+ :
398.1863. Found 398.1869.
10b: 66%; 1H NMR (400 MHz, CDCl3) δ 7.45 (1 H, s), 6.92 (1 H, s), 6.88 (1 H, dd, J = 1.4, 4.0 Hz),
6.21 (1 H, s), 6.17 (1 H, dd, J = 1.4, 4.0 Hz), 3.91 (3 H, s), 3.57 (3 H, s), 2.40 (3 H, s); 13C NMR
(150 MHz, CDCl3) δ 160.7, 160.2, 155.9, 152.1, 135.3, 128.0, 119.2, 117.4, 114.1, 113.5, 112.8,
110.7, 105.5, 99.6, 56.2, 33.5, 18.7; HRMS (ESI+) Calcd. for C17H15N2O3 [M + H]+ : 295.1077.
Found 295.1077.
10c: 29%; 1H NMR (400 MHz, CDCl3) δ 7.62 (1 H, s), 7.21 (1 H, s), 7.05 (1 H, d, J = 3.6 Hz), 6.28
(1 H, d, J = 1.2 Hz), 6.22 (1 H, d, J = 3.6 Hz,), 3.87 (3 H, s), 3.86 (2 H, q, J = 7.2 Hz), 2.40 (3 H, d, J
= 0.8 Hz), 1.18 (3 H, t, J = 6.8 Hz); 13C NMR (150 MHz, CDCl3) δ 160.6, 160.2, 155.9, 152.1, 134.2,
128.1, 119.8, 117.5, 114.1, 113.5, 112.8, 110.9, 103.9, 99.6, 56.2, 41.9, 18.7, 16.3; HRMS (ESI+)
Calcd. for C18H17N2O3 [M + H]+ : 309.1234. Found 309.1231.
10d: 40%; 1H NMR (400 MHz, CDCl3) δ7.43 (1 H, s), 6.91 (1 H, s), 6.88(1 H, d, J = 4.1 Hz), 6.21(1
H, s), 6.13 (1 H, d, J = 4.1 Hz), 3.89-.386 (5 H, m), 2.40 (3 H, s), 1.61 (2 H, t, J = 7.3 Hz), 1.18-1.09
(6 H, m), 0.77 (3 H, t, J = 6.9 Hz); 13C NMR (150 MHz, CDCl3) δ 160.9, 160.3, 155.9, 152.3, 134.6,
128.2, 119.8, 117.8, 114.4, 113.6, 112.9, 111.0, 104.6, 99.7, 56.3, 47.1, 31.2, 31.0, 26.2, 22.6, 18.9,
14.0; HRMS (ESI+) Calcd. for C22H25N2O3 [M + H]+ : 365.1860. Found 365.1850.
10e: 38%; 1H NMR (400 MHz, CDCl3) δ 7.40(1 H, s), 6.89(1 H, s), 6.86(1 H, d, J = 4.0 Hz), 6.20(1
H, s), 6.10(1 H, d, J = 3.6 Hz), 3.86(3 H, s), 3.71(2 H, d, J = 7.6 Hz), 2.38(3 H, s), 1.55-1.59(4 H, m),
1.35-1.38(2 H, m), 0.99-1.09(3 H, m), 0.66-0.75(2 H, m); 13C NMR (150 MHz, DMSO-d6) δ 159.9,
159.8, 155.2, 153.3, 135.1, 128.4, 119.5, 116.9, 114.3, 112.9, 111.8, 111.3, 103.8, 99.9, 56.5, 52.3,
29.7, 25.5, 25.0, 18.2; HRMS (ESI+) Calcd. for C23H25N2O3 [M + H]+ : 377.1860. Found 377.1865.
10f: 33%; 1H NMR (400 MHz, CDCl3) δ7.23-7.20 (3 H, m), 7.18 (1 H, s), 6.96(1 H, d, J = 4.0 Hz),
6.87-6.85 (2 H, m), 6.85 (1 H, s), 6.23 (1 H, d, J = 3.6 Hz), 6.14 (1 H, d, J = 1.6 Hz), 5.09 (2 H, s),
3.77 (3 H, s), 2.16 (3 H, d, J = 1.2 Hz); 13C NMR (150 MHz, CDCl3) δ 160.6, 160.2, 155.8, 152.1,
136.6, 134.8, 128.6, 128.2, 127.8, 126.5, 119.9, 117.2, 114.0, 113.2, 112.6, 111.5, 105.3, 99.4, 56.1,
50.3, 18.4; HRMS (ESI+) Calcd. for C23H19N2O3 [M + H]+ : 371.1390. Found 371.1400.

10g: 22%; 1H NMR (400 MHz, CD3OD) δ 7.62 (2 H, d, J = 8.4 Hz), 7.45 (1 H, s), 7.06(2 H, d, J =
8.4 Hz), 7.04 (1 H, d, J = 4.0 Hz), 7.02 (1 H, s), 6.32 (1 H, d, J = 4.0 Hz), 6.18 (1 H, d, J = 0.8 Hz),
5.25 (2 H, s), 3.76 (3 H, s), 2.31 (3 H, d, J = 0.8 Hz); 13C NMR (150 MHz, CDCl3) δ 160.6, 160.1,
156.1, 151.9, 141.9, 135.3, 132.6, 128.3, 127.3, 120.7, 118.4, 116.8, 113.8, 113.6, 113.1, 112.0, 111.9,
105.4, 99.8, 56.2, 49.9, 18.7; HRMS (ESI+) Calcd. for C24H18N3O3 [M + H]+ : 396.1343. Found
396.1332.
10h: 44%; 1H NMR (400 MHz, CDCl3) δ6.95-6.87 (7 H, m), 6.22-6.17 (2 H, m), 5.06(2 H, s), 3.78
(3 H, s), 2.23 (3 H, s); 13C NMR (150 MHz, CDCl3) δ 163.1, 161.5, 160.7, 160.2, 155.9, 152.2,
134.9, 132.5, 132.4, 128.6, 128.5, 128.3, 120.3, 117.2, 115.8, 115.6, 114.1, 113.5, 112.9, 111.7, 105.2,
99.6, 56.3, 49.8, 18.6; HRMS (ESI+) Calcd. for C23H18FN2O3 [M + H]+ : 389.1296. Found 389.1291.
4.2.13. Synthesis of 5-(5-cyano-1-methyl-2-pyrrolyl)-6-hydroxycoumarin (11)
Boron tribromide (1 M solution in dichloromethane, 4 mL) was added to a solution of 10b (48.6 mg,
0.17 mmol) in dichloromethane (6.0 mL) at –78C under argon. The mixture was stirred at room
temperature for 2 d, then poured into water, and extracted with ethyl acetate. The organic layer was
washed with water and brine, dried over magnesium sulfate, filtered, and concentrated. The residue
was purified by column chromatography (silica gel, AcOEt / n-hexane = 1/2) to give 11 (21.1 mg,
44%). 1H NMR (400 MHz, CDCl3) δ 11.13 (1 H, s), 7.58 (1 H, d, J = 1.6 Hz), 7.03 (1 H, dd, J = 2.0,
4.4 Hz), 6.89 (1 H, d, J = 1.6 Hz), 6.27 (1 H, dd, J = 2.0, 4.0 Hz), 6.20 (1 H, s), 3.56 (3 H, s), 2.38 (3
H, s); 13C NMR (150 MHz, CDCl3) δ 160.0, 158.9, 154.9, 153.5, 136.1, 128.9, 119.3, 115.7, 114.2,
112.2, 111.0, 110.8, 103.8, 102.5, 33.5, 18.1; HRMS (ESI+) Calcd. for C16H12N2NaO3 [M + Na]+ :
303.0740. Found 303.0739.
4.3 Biology
4.3.1. Alkaline phosphatase assay
Alkaline phosphatase assay was performed as reported.22 T47D breast-carcinoma cells were
cultured in RPMI 1640 medium with 10% (v/v) fetal bovine serum. Cells were plated in 96-well
plates at 104 cell/well and incubated overnight (37°C, 5% CO2 in air). The next day, cells were
treated with fresh medium containing test compound, and further incubated for 24 h. The medium
was aspirated and the cells were fixed with 100 L of 1.8% formalin (in PBS). The fixed cells were
washed with PBS and 75 L of assay buffer (1 mg/mL p-nitrophenol phosphate in diethanolamine
water solution, pH 9.0, 2 mM MgCl2) was added. The mixture was incubated at room temperature
with shielding from light for 2 h, and then the reaction was terminated by the addition of 100 L of 1
M NaOH. The absorbance at 405 nm was measured using a plate reader.

4.3.2. hPR-binding assay
PR-binding experiments were performed as reported, using recombinant hPR-LBD purchased from
Invitrogen.22 hPR-LBD was diluted with buffer (20 mM Tris.HCl, 300 mM NaCl, 1 mM EDTA, 5
mM DTT, pH 8.0) to 5 nM and 300 L aliquots were incubated in the dark at 4°C with 4 nM
[1,2,6,7-3H]progesterone (Perkin Elmer) and reference or test compounds (dissolved in DMSO; final
concentration of DMSO was 3%). Nonspecific binding was assessed by addition of a 200-fold
excess of nonradioactive progesterone. After 24 h, 30 L of Dextran T-70/c-globulin-coated charcoal
suspension was added to the ligand/protein mixtures (1% activated charcoal, 0.05% -globulin,
0.05% Dextran 70, final concentrations) and incubated at 4°C for 5 min. The charcoal was removed
by centrifugation for 5 min at 1300 g, and the radioactivity of the supernatant was measured in
Ultima Gold scintillation cocktail (Perkin Elmer) by using a liquid scintillation counter. All
experiments were performed in duplicate.
4.3.3. SC-3 Growth Inhibition Assay
SC-3 Growth inhibition assay was performed as reported.22,26 SC-3 cells were cultured in MEMα
(Wako Co.) supplemented with 2% FBS and 1 nM DHT at 37°C under 5% CO2. All experiments
were performed in triplicate or more. Cells were trypsinized and diluted to 20,000 cells/mL with
MEMα supplemented with 2% charcoal-stripped FBS. This cell suspension was seeded in 96-well
plates at a volume of 100 μL and incubated at 24 h. After removal of 10 μL of medium from each
well, 10 μL of the drug solution, supplemented with serial dilutions of the test compounds or DMSO
as a dilution control in the presence of 1 nM DHT, was added. Then the plates were incubated at
37°C under 5% CO2 for 3 days, and the cell number was determined using a Cell Counting Kit-8
(Dojindo). A 10 μL aliquot of WST-8 was added to each well of the microcultures, and the cells were
incubated for 2 h. The absorbance at 450 nm was measured with a microplate reader. This parameter
is related to the number of living cells in the culture.
4.3.4. hGR-binding assay
hGR-binding experiments were performed at Caliper Life Science (US), according to the reported
method.22 The hGR was diluted with binding buffer (50 mM KH2PO4 pH 7.4 with 10 mM sodium
molybdate and 1 mM ditiothreitol) to yield a final protein concentration of 1.25 nM in the assay
tubes. The final incubation conditions were: [3H]dexamethasone, 10-9 M; triamcinolone acetonide,
10-5 M (for non-specific binding determination only); test compounds, 10-7 to 10-5 M, 0.4% DMSO.
After 18 h, the bound ligand was assayed by vacuum filtration onto glass fiber filters and
radioactivity was counted in 50 μl of scintillation cocktail (Microscint-20). All experiments were
performed in duplicate.

4.3.5. rMR-binding assay
rMR-binding experiments were performed at Caliper Life Science (US) , according to the reported
method.22 The rMR was diluted with binding buffer (20 mM HEPES pH 7.4, 15 mM sodium
molybdate, 1 mM dithiothreitol, 10% glycerol and 1 mM EDTA). The concentration for the MR
receptor tissue preparation was optimized for each tissue preparation, and was 16.7 mg wet
weight/mL in the final reaction for the present assays. The final incubation conditions were:
[3H]aldosterone, 2 × 10-9 M; spironolactone, 10-6 M (for non-specific binding determination only);
test compounds, 10-7 to 10-5 M; 0.4% DMSO. After 18-20 h, the unbound ligand was removed by
absorption on dextran-coated charcoal, followed by centrifugation. The radioactivity of bound ligand
in 200 μl of the supernatant was counted in 6 mL of scintillation cocktail (Luma Safe). All
experiments were performed in duplicate.
4. 4. Fluorescence study
Fluorescence spectra were recorded with a JASCO FP-6600. Fluorescence quantum yields (Φfl) were
determined using quinine sulfate (0.577) in 0.1 M H2SO4 as a standard. Fluorescence spectra in the
presence of PR-LBD were measured in the same buffer as that used for hPR binding assay (50 mM
KH2PO4 pH 7.4 with 10 mM sodium molybdate and 1 mM dithiothreitol).
Acknowledgments
This work was partly supported by JSPS KAKENHI Grant No 16K08318 (to AT), No 15K08019 (to
TH), No. 25460146 (to SF), JSPS Core-to-Core Program, A. Advanced Research Networks, and
MEXT Platform for Drug Discovery, Informatics, and Structural Life Science, Japan. TH and AT
gratefully acknowledge funding from Terumo Life Science Foundation. A.T. also thanks The Naito
Foundation.
References
1) Psychoyos, A. Vitam. Horm. 1973, 31, 201.
2) Zhang, Z.; Funk, C.; Glasser, S. R.; Mulholland, J. Endocrinology 1994, 135, 1256.
3) Mani, S. K.; Allen, J. M. C.; Clark, J. H.; Blaustein, J. D.;O’Malley, B. W. Science 1994, 265,
1246.
4) Lydon, J. P.; Demayo, F. J.; Funk, C. R.; Mani, S. K.; Hughes, A. R.; Montgomery, C. A.;
Shyamala, G.; Conneely, O. M.; O’Malley, B. W. Gene Dev. 1995, 9, 2266.
5) Simoncini, T.; Mannella, P.; Formari, L.; Caruso, A.; Varone, G.; Genazzani, A. R. Steroids 2003,
68, 831.
6) Szekeres-Bartho, J.; Polgar, B.; Kozma, N.; Miko, E.; Par, G.; Szereday, L.; Barakonyi, A.;

Palkovics, T.; Papp, O.; Varga, P. Chem. Immunol. Allergy 2005, 89, 118
7) Singh, M. Ann. N. Y. Acad. Sci. 2005, 1052. 145.
8) Laudet V.; Gronemeyer H. The Nuclear Receptor FactsBook, Academic Press, London, 2001.
9) Spitz I. M. Progesterone antagonists and progesterone receptor modulators: an overview. Steroids,
2003, 68, 981.
10) Winneker R. C.; Fensome A.; Zhang P.; Yudt M. R.; McComas C. C.; Unwalla, R. J. A. Steroids
2008, 73, 689.
11) Teutsch, G.; Philibert, D. History and perspectives of anti-progestins from the chemist’s point of
view. In Human Reproduction; Edwards, R. G., Ed.; Oxford University Press: Oxford, England,
1994; Vol. 9, p 12. Suppl. 1.
12) Kettel, L. M.; Murphy, A. A.; Mortola, J. F.; Liu, J. H.; Ulmann, A.; Yen, S. S. Fertil. Steril. 1991,
56, 402.
13) Murphy, A. A.; Kettel, L. M.; Morales, A. J.; Roberts, V. J.; Yen, S. S. J. Clin. Endocrinol. Metab.
1993, 76, 513.
14) Kettel, L. M. Clin. Obstet. Gynecol. 1995, 38, 921.
15) Chabbert-Buffet, N.; Meduri, G.; Bouchard, P.; Spitz, I. M. Hum. Reprod. Update 2005, 11, 293.
16) Madauss, K. P.; Stewart, E. L.; Williams, S. P. Med. Res. Rev. 2007, 27, 374.
17) Zhang, P.; Terefenko, E. A.; Fensome, A.; Wrobel, J.; Winneker, R.; Lundeen, S.; Marschke, K.
B.; Zhang, Z. J. Med. Chem. 2002, 45, 4379.
18) Collins, M. A.; Hudak, V.; Bender, R.; Fensome, A.; Zhang, P.; Miller, L.; Winneker, R. C.;
Zhang, Z.; Zhu, Y.; Cohen, J.; Unwalla, R. J.; Wrobel, J. Bioorg. Med. Chem. Lett. 2004, 14, 2185.
19) Fensome, A.; Bender, R.; Chopra, R.; Cohen, J.; Collins, M. A.; Hudak, V.; Malakian, K.;
Lockhead, S.; Olland, A.; Svenson, K.; Terefenko, E. A.; Unwalla, R. J.; Wilhelm, J. M.; Wolfrom,
S.; Zhu, Y.; Zhang, Z.; Zhang, P.; Winneker, R. C.; Wrobel, J. J. Med. Chem. 2005, 48, 5092.
20) Terefenko, E. A.; Kern, J.; Fensome, A.; Wrobel, J.; Zhu, Y.; Cohen, J.; Winneker, R.; Zhang, Z.;
Zhang, P. Bioorg. Med. Chem. Lett. 2005, 15, 3600.
21) Fensome, A.; Adams, W. R.; Adams, A. L.; Berrodin, T. J.; Cohen, J.; Huselton, C.; Illenberger,
A.; Kern, J. C.; Hudak, V. A.; Marella, M. A.; Melenski, E. G.; McComas, C. C.; Mugford, C. A.;
Slayden, O. D.; Yudt, M.; Zhang, Z.; Zhang, P.; Zhu, Y.; Winneker, R. C.; Wrobel, J. E. J. Med.
Chem. 2008, 51, 1861.
22) Sakai, H.; Hirano, T.; Mori,S.; Fujii, S.; Masuno, H.; Kinoshita, M.; Kagechika, H.; Tanatani, A.
J. Med. Chem. 2011, 54, 7055.
23) Shiraishi, T.; Kagechika, H.; Hirano, T. New J. Chem. 2015, 39, 8389.
24) Hirano, T.; Hiromoto, K.; Kagechika, H. Org. Lett. 2007, 9, 1315.
25) Still, W. C.; Gennari, C. Tetrahedron Lett. 1983, 41, 4405.
26) Hiraoka, D.; Nakamura, N.; Nishizawa, Y.; Uchida, N.; Noguchi, S.; Matsumoto, K.; Sato, B.

Cancer Res. 1987, 47, 6560.

Graphical Abstract
Development of 6-Arylcoumarins as Nonsteroidal Progesterone Antagonists.
Structure-activity Relationships and Fluorescence Properties
Marie Kinoshita, Mai Negishi, Haruka Sakai, Tomoya Hirano, Shuichi Mori, Shinya
Fujii, Hiroyuki Kagechika, and Aya Tanatani,